Registration Dossier

Administrative data

Endpoint:
neurotoxicity: oral
Remarks:
other: Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted on structural analogue and suitable for read across. Guideline GLP study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Neurobehavioral evaluation added to protocol.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Phosphorodithioic Acid, Mixed O,O-Bis(Iso-Bu and Pentyl) Esters, Zinc Salts
- Substance type: Brown, translucent viscous liquid
- Analytical purity: Considered to be 100.0%. Documentation regarding the purity and stability of the test item is on file with the Sponsor and WIL.
- Lot/batch No.: 1039274
- Stability under test conditions: stable
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
Each rat was uniquely identified by a Monel® metal ear tag displaying the animal number.

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Raleigh, NC
- Age at study initiation: approximately 11 weeks old at initiation of treatment; the 10 rats/sex/group used for pairing were approximately 13 weeks old when paired on study day 13.
- Weight at study initiation: 335 g to 429 g (males) and 227 g to 279 g (females); female body weights ranged from 238 g to 319 g on gestation day 0.
- Housing: Following receipt and until pairing for 10 animals/sex/group, all animals were housed individually in clean, stainless steel wire mesh cages suspended above cage board. The cage-board was changed at least 3 times per week. During cohabitation, rats were paired for mating in the home cage of the male. Following positive evidence of mating, the males were housed in suspended wire mesh cages until the scheduled necropsy, and the females were transferred to plastic maternity cages with nesting material, ground corncob bedding (Bed O'Cobs®; The Andersons, Cob Products Division, Maumee, OH). The dams and their litters were housed in these cages until euthanasia on lactation day 4. Females that failed to deliver were housed in plastic maternity cages until post-mating day 25. Males and females not used for pairing remained in suspended stainless steel wire mesh cages until euthanasia.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 ad libitum except during the period of fasting of all males and post-treatment phase females prior to clinical pathology blood collection Feeders were changed and sanitized once per week.
- Water: Reverse osmosis-purified (on site) drinking water, delivered by an automatic watering system ad libitum
- Acclimation period: 13 days prior to the first day of treatment. During the acclimation period, the animals were observed twice daily for mortality and general changes in appearance and behavior.

ENVIRONMENTAL CONDITIONS
- Temperature: 71.1 to 72.3 deg F (21.7 to 22.4 deg C)
- Humidity: 36.3% to 63.6%
- Air changes: 10 fresh air changes per hour
- Photoperiod: 12 hour light (0600 hours to 1800 hours)/12 hour dark photoperiod

IN-LIFE DATES: From:8 December 2009 To: 12 February 2010 (Last female necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Mineral Oil USP
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item formulations were prepared approximately weekly as single formulations for each dosage level, divided into aliquots for daily dispensation, and stored at room temperature. The test item formulations were stirred continuously throughout the preparation, sampling, and dose administration procedures. The vehicle and test item formulations were administered orally by gavage, via an appropriately sized flexible, Teflon®-shafted, stainless steel ball-tipped dosing cannula (Natume, Japan) once daily.

VEHICLE
- mineral oil, USP
- Supplier: Spectrum Chemical Manufacturing Corporation, New Brunswick, NJ and Gardena, CA locations.
- The vehicle was dispensed into glass container approximately weekly for administration to the control group (Group 1) and for preparation of the test item formulations; aliquots were prepared for daily dispensation to the control group and stored at room temperature. The vehicle was mixed throughout preparation, sampling, and dose administration procedures.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the formulated test item, at concentrations between 0.5 and 20 mg/mL in mineral oil, USP, was established by the Sponsor. Formulations are stable after 10 and 30 days of stationary storage under room temperature conditions. Therefore, stability analyses were not conducted as part of this study.
Quadruplicate samples for homogeneity and concentration analyses were collected from the middle of the vehicle formulation and from the top, middle, and bottom strata of each test item batch formulation. One set of duplicate samples from each batch was shipped under ambient conditions to the sponsor for dose formulation analyses. The remaining set of duplicate samples was stored under ambient conditions as back-up. The analyzed dosing formulations were within WIL Research’s SOP range for solutions (90% to 110%) and were homogeneous.
Duration of treatment / exposure:
Males: 10/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses.
Females: 10/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 40-52 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses.

The extra 5 males and 5 females in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post treatment period and remained on study for a 14-day non-dosing period. These animals were not evaluated for reproductive parameters.
Frequency of treatment:
Once daily at approximately the same time each day.

Doses / concentrations
Remarks:
Doses / Concentrations:
0, 10, 40, or 160 mg/kg/day administered at a dosage volume of 10 mL/kg.
Basis:
actual ingested
gavage
No. of animals per sex per dose:
The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. Concurrent control group of 15 rats/sex



Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosage levels were determined from the results of previous studies conducted
- Rationale for animal assignment: body weight stratification in a block design using a computer randomization procedure


Examinations

Observations and clinical examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All rats were observed twice daily, once in the morning and once in the afternoon, for moribundity and mortality. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

DETAILED CLINICAL OBSERVATIONS: Yes, A detailed physical examination was conducted weekly on each animal beginning approximately 3 days prior to the initiation of dose administration. Each male and female was also observed for signs of toxicity approximately 1 hour following dose administration. The absence or presence of findings was recorded for individual animals. In addition, the presence of findings at the time of dose administration was recorded for individual animals. Mated females expected to deliver were also observed twice daily during the period of expected parturition and at parturition for dystocia (prolonged labor, delayed labor) or other difficulties.

CLINICAL PATHOLOGY: Yes, Blood samples for clinical pathology evaluations (hematology and serum chemistry) were collected from 5 animals/sex/group at the scheduled necropsies (study day 28 for males selected for breeding and lactation day 4 for females) and from 5 animals/sex in the control and high-dose groups following a 14-day non-dosing post-treatment period (study day 42 for males and study 53 for females). All males (including those not scheduled for clinical pathology assessments) and the post-treatment phase females were fasted overnight prior to blood collection with water available. Blood for serum chemistry and hematology was collected from the retro orbital sinus following isoflurane anesthesia. Blood for coagulation parameters was collected from the vena cava at the time of necropsy. Blood was collected into tubes containing EDTA (hematology), sodium citrate (clotting determinations), or no anticoagulant (serum chemistry).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual male body weights were recorded 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly throughout the study and prior to the scheduled euthanasia. Individual female body weights were recorded beginning approximately 1 week prior to the initiation of dose administration, on the first day of dose administration, and weekly thereafter until evidence of copulation was observed for females selected for pairing. Once evidence of mating was observed, female body weights were recorded on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 0 (when possible), 1, and 4.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Individual food consumption was recorded for both males and females on the corresponding weekly body weight days until pairing. Food consumption continued to be recorded for males and females not selected for pairing until euthanasia. For animals selected for paiting, once evidence of mating was observed, food consumption was recorded for females on gestation days 0, 4, 7, 11, 14, 17, and 20 and on lactation days 1 and 4.
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes, FOB assessments were recorded for 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females).
- Parameters examined: Posture, Convulsions/Tremors, Feces consistency, Biting, Palpebral (eyelid) closure, Ease of removal from cage, Lacrimation/Chromodacryorrhea, Piloerection, Palpebral closure, Eye prominence, Red/Crusty deposits, Ease of handling animal in hand, Salivation, Fur appearance, Respiratory rate/character, Mucous membranes/Eye/Skin color, Muscle tone, Mobility, Rearing, Convulsions/Tremors, Grooming, Bizarre/Stereotypic behavior, Time to first step (seconds), Gait, Arousal, Urination/Defecation, Gait score, Backing (Note: Open field observations were evaluated over a 2-minute observation period.), Approach response, Startle response, Pupil response, Forelimb extension, Air righting reflex, Touch response, Tail pinch response, Eyeblink response, Hindlimb extension, Olfactory orientation, Hindlimb extensor strength, Hindlimb foot splay, Grip strength hind and forelimb, Rotarod performance
- Description of procedures: The FOB used at WIL is based on previously developed protocols (Gad, 1982; Haggerty, 1989; Irwin, 1968; Moser et al., 1988, Moser et al., 1991, and O’Donoghue, 1989).
- Technicians were blind to treatment status of animals: Yes
- Site of testing: sound attenuated room equipped with a white noise generator set to operate at 70 ± 10 dB.

LOCOMOTOR ACTIVITY: Yes, 5 animals/sex/group prior to dose administration on study day 27 (males selected for pairing) and on lactation day 4 (females); the same animals evaluated for FOB were selected for locomotor activity assessment.
- Replicates used: yes
- Type of equipment used: Locomotor activity, recorded after the completion of the FOB, was measured automatically using a personal computer controlled system utilizes a series of infrared photobeams surrounding a clear plastic, rectangular cage to quantify each animal’s motor activity. Four-sided black plastic enclosures were used to surround the clear plastic boxes to decrease the potential for distraction from extraneous environmental stimuli or stimuli from biologists or adjacent animals. The black enclosures rested on top of the photobeam frame and did not interfere with the path of the beams.
- Total activity: Data for ambulatory and total motor activity were tabulated. Total motor activity was defined as a combination of fine motor skills (i.e., grooming, interruption of 1 photobeam) and ambulatory motor activity (interruption of 2 or more consecutive photobeams).
- Other: Each animal was tested separately. Data were collected in 5 minute epochs and the test session duration was 60 minutes. These data were compiled as six, 10-minute subintervals for tabulation.

Sacrifice and (histo)pathology:
SACRIFICE
- All animals were euthanized by carbon dioxide inhalation. Males selected for pairing were euthanized following completion of the mating period. Males not selected for pairing were euthanized following the 14-day non-dosing post-treatment period. Females that delivered were euthanized on lactation day 4. Females (with evidence of mating) that failed to deliver were euthanized on post mating day 25.

GROSS NECROPSY
- A complete necropsy was conducted on animals euthanized in extremis or at the scheduled necropsies. Necropsy included examination of the external surface, all orifices, the cranial cavity, the external surface of the brain, and the thoracic, abdominal, and pelvic cavities, including viscera.

HISTOPATHOLOGY
- The following tissues were examined microscopically from all treatment phase animals in the control and high dose groups and from the high dose male that was euthanized in extremis. Adrenal glands (2), Aorta, Bone with marrow (sternebrae), Bone marrow smeara, Brain, Cerebrum level 1, Cerebrum level 2, Cerebellum with medul/pons, Coagulating gland, Eyes with optic nerve (2)b, Gastrointestinal tract, Esophagus, Stomach, Duodenum, Jejunum, Ileum, Cecum, Colon, Rectum, Heart, Kidneys (2), Liver (sections of 2 lobes), Lungs (including bronchi, fixed by inflation with fixative), Lymph node (Axillary, Mesenteric, Mandibular), Ovaries and oviducts (2), Pancreas, Peripheral nerve (sciatic), Pituitary gland, Prostate gland, Mandibular salivary glands (2), Seminal vesicles (2), Skeletal muscle (rectus femoris), Skin with mammary glandc, Spinal cord (cervical), Spleen, Testes with epididymidesd (2), Thymus, Thyroids [with parathyroids, if present (2)], Trachea, Urinary bladder, Uteruse with cervix and vagina, All gross lesions (all groups)
In addition, the non-glandular portion of the stomach, thymus, and all gross lesions (all animals) and the testes at all dosage levels were examined microscopically at the scheduled necropsies for both treatment and post treatment phase animals.

ORGAN WEIGHTS
- The following organs were weighed from all F0 animals at the scheduled necropsies: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries with oviducts, Spleen, Testes, Thymus gland, Thyroids with parathyroids.
Statistics:
Each mean was presented with the standard deviation (S.D.), standard error (S.E.), and the number of animals (N) used to calculate the mean. Due to the use of significant figures and the different rounding conventions inherent in the types of software used, the means and standard deviations on the summary and individual tables may differ slightly. Data obtained from nongravid females were excluded from statistical analyses following the mating period. Where applicable, the litter was used as the experimental unit.
All statistical tests were performed using WTDMS™ unless otherwise noted. Analyses were conducted using two-tailed tests (except as noted otherwise) for minimum significance levels of 1% and 5%, comparing each test item-treated group to the control group by sex.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
no effects observed
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

VIABILITY (OFFSPRING)
Mean numbers of corpora lutea and unaccounted-for sites, mean number of pups born, live litter size, the percentage of males at birth, and postnatal survival in the 10, 40, and 160 mg/kg/day groups were similar to the control group values.

CLINICAL SIGNS (OFFSPRING)
The general physical condition of all F1 pups in this study were unaffected by test item administration. No test item-related clinical findings were noted for the F1 pups.

BODY WEIGHT (OFFSPRING)
Mean male and female pup body weights and body weight gains in the 10, 40, and 160 mg/kg/day groups were unaffected by test item administration during PND 1-4. No statistically significant differences from the control group were noted.

GROSS PATHOLOGY (OFFSPRING)
There were no remarkable macroscopic findings in the F1 pups at the scheduled necropsy at any dosage level. (migrated information)
Details on results:
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One male in the 160 mg/kg/day group was euthanized in extremis on study day 17; a gross observation of a thickened stomach was noted at necropsy. Clinical findings noted for this male approximately 1 hour following dose administration on the day of euthanasia consisted of yellow material on various body surfaces, clear material around the mouth, unkempt appearance, decreased defecation, and labored respiration. Microscopically, this male was noted with inflammation, edema, and ulceration in the non-glandular stomach, erosion and inflammation in the trachea, and lymphoid depletion in the thymus, spleen, and lymph nodes (mesenteric, mandibular, and axillary). The lesions in the non glandular stomach were considered test item-related and may have contributed to the moribund state of this male. All other animals in all dosage groups survived to the scheduled necropsies.
Test item related clinical findings were noted in the 160 mg/kg/day group males and females and included rales, decreased, shallow, and/or labored respiration and salivation related findings. These findings were noted at the daily examinations, at the time of dosing, and/or approximately 1 hour following dose administration primarily during the treatment period. However, because of their sporadic occurrence, these cardio pulmonary findings were considered to be incidental and secondary to the nature of the test item and the route of administration.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Mean body weights, body weight changes, and food consumption were unaffected by test item administration in the 10, 40, and 160 mg/kg/day groups throughout the treatment and post-treatment periods.

FUNCTIONAL OBSERVATIONAL BATTERY (FOB)
HOME CAGE OBSERVATIONS
Home cage parameters were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

HANDLING OBSERVATIONS
Handling parameters were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females). Male no. 61749 in the 160 mg/kg/day group was noted with rales at the handling observations on study day 27. This finding was consistent with the test item-related cardio-pulmonary-related clinical findings noted at the daily observations.

OPEN FIELD OBSERVATIONS
Open field parameters were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

SENSORY OBSERVATIONS
Sensory parameters were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

NEUROMUSCULAR OBSERVATIONS
Neuromuscular parameters were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

PHYSIOLOGICAL OBSERVATIONS
Physiological parameters (catalepsy, body temperature, and body weight) were unaffected by test item administration. There were no statistically significant differences for the test item treated groups when compared to the control group on study day 27 (males) or lactation day 4 (females).

LOCOMOTOR ACTIVITY (PARENTAL ANIMALS)
Locomotor activity patterns (total activity as well as ambulatory activity counts) were unaffected by test substance administration at all concentrations when evaluated on study day 27 (males) and lactation day 4 (females). Values obtained from the 6 subintervals evaluated (0-10, 11-20, 21-30, 31-40, 41-50, and 51-60 minutes) and the overall 60 minute test session values were comparable to the concurrent control values and the WIL historical control data. Differences from the control group were slight, not statistically significant when analyzed by a repeated measures analysis, within the WIL historical control data ranges and/or did not occur in a dose/exposure-related manner. Mean total and ambulatory activity counts noted for males in the 160 mg/kg/day group were 23.2% to 33.9% lower than the control group during the 11-20 and 21-30 minute subintervals. These decreases were attributed to greater habituation in these animals compared to the control group animals, and unrelated to test item administration.

CLINICAL PATHOLOGY (PARENTAL ANIMALS)
There were no test substance-related effects on serum chemistry, hematology, or coagulation parameters in the 10, 40, and 160 mg/kg/day groups.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Male and female mating and fertility, male copulation and female conception indices, mean number of days between pairing and coitus, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There were no test item-related alterations in final body weight or organ weights at any dosage level. Significant (p<0.05) differences were observed when the control and high dose group males were compared at the recovery (post-treatment) necropsy and consisted of lower mean kidney weight relative to body weight, higher mean spleen weight relative to brain weight, higher mean left testis weight relative to brain weight, and higher mean right testis weights (absolute and relative to brain weight). There was no case where all 3 measures (absolute, relative to body weight, and relative to brain weight) were statistically significant. Thus, since the absolute weights and weights relative to body or brain weight were discordant, these organ weight changes were considered to be spurious.

GROSS PATHOLOGY (PARENTAL ANIMALS)
One male (no. 61739) in the 160 mg/kg/day group was euthanized in extremis on study day 17. The thickened stomach noted macroscopically for this male was related to test item administration. There were no other test item related internal findings observed for either sex at any dosage level at the scheduled necropsies. Macroscopic findings observed in the test item groups occurred infrequently and/or in a manner that was not dose related.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Test item-related histologic observations of epithelial hyperplasia, hyperkeratosis, and inflammation of the non-glandular stomach, typically at the limiting ridge, but sometimes more widespread, were noted in the 160 mg/kg/day group males and females.

Effect levels

open allclose all
Dose descriptor:
NOEL
Remarks:
portal-of-entry
Effect level:
40 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Localized injury to the nonglandular portion of the stomach.
Remarks on result:
other: Generation:
Dose descriptor:
NOAEL
Remarks:
systemic toxicity, including neurobehavioral
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects observed at any dose level.
Remarks on result:
other: Generation:
Dose descriptor:
NOEL
Remarks:
neonatal toxicity
Effect level:
160 mg/kg bw/day (actual dose received)
Based on:
other: maternal dose
Sex:
male/female
Basis for effect level:
other: No effects observed at any dose level.
Remarks on result:
other: Generation: offspring (migrated information)

Any other information on results incl. tables

JUSTIFICATIONS FOR READ-ACROSS

EC 283-392-8 has not been tested for repeat dose toxicity, however experimental data on structurally related substances EC 270-608-0 (OECD 422) is available and suitable for read-across. Justifications for read-across:

Manufacture/Usage:

EC 283-392-8 and EC 270-608-0 are generically referred to as zinc dialkylthiophosphate (ZDDP) which are produced under similar manufacturing procedures and used in commerce as multi-functional anti-wear and anti-oxidation inhibitor performance components in passenger motor oils, diesel engine oils and industrial oils such as hydraulic lubricants.

Chemical Similarity:

EC 283-392-8 and EC 270-608-0 consist of alkyl substituted phosphorodithioic acid structures complexed with zinc.

EC 283-392-8:phosphorodithioic acid, mixed O,O-bis(1,3-dimethylbutyl and iso-propyl) esters, zinc salts, referred to as “mixed isopropyl and 1,3-dimethylbutyl derivative”

EC 270-608-0:phosphorodithioic acid, mixed O,O-bis(iso-butyl and pentyl) esters, zinc salts – (CAS 68457-79-4), referred to as “mixed isobutyl and pentyl derivative”

These ZDDPs share similar core structures - alcohol ester of dithiophosphate, specific structural variations that relate to their alcohol group substituent are the alkyl chain length and the degree of branching of the alcohol charge.

Using Tanimoto Fingerprint (ToxMatch Version 1.06 software) to model chemical structures of the analogues showed comparable values for relevant molecular descriptors (e.g., number of H bond acceptor atoms), and gave a similarity index greater than 0.8 (values range from 0, no similarity to 1, identical; peer reviewed literature indicates that values greater than 0.6 are significantly similar); therefore chemical structures of the analogues have determined to be sufficiently close for there to be a reasonable expectation of similar toxicological effects.

Physicochemical Properties:

Standard physicochemical properties for each substance were listed in Table 1.

Table 1. Establishment of similarity between the data donating substance and the data accepting substance

EC

Alkyl Group

MW

Log Kow

Water Sol (ppm)

Vapor Pressure (Pa at 25 oC)

283-392-8

(accept data)

C3/C6, branched

578

0.56

2764

6.9 x10-3

270-608-0

(donate data)

C4, branched

C5, mixture of linear and branched

578

0.69

1658

2.5 x10-3

As shown in Table 1, these two substances have the same molecular weight, similar Log Kow, low vapor pressure, and both are water soluble. The similarity of the physicochemical properties of these substances parallels their structural similarity.

Biologically Active Functional Groups:

The ester group presents in each of the analogue members, and is expected to exhibit similar biological activities. Non-random patterns were observed for the toxicological effects (e.g. available data showed low levels of acute toxicity effect, lacking of mutagenic effect in bacteria, consistent trend of change in ecotoxicity effect, etc.), these common behaviors and consistent trends suggest a common mechanism/mode of action, with little influence from the length of carbon chain. These facts further supported read-across between the analogue members.

Available Data and Adequacy for Read-across:

EC 270-608-0 was evaluated for systemic toxicity (including neurobehavioral parameters) with rats at doses as high as 160 mg/kg/day for up to 52 consecutive days in accordance with OECD 422. Under the conditions of this screening study, no test material-related effects on neurobehavioral parameters were noted at any dosage level. Substance-related toxicity was limited to morbundity, adverse clinical signs, and epithelial hyperplasia, hyperkeratosis, and inflammation of the stomach. The parental NOAEL for systemic toxicity was 160 mg/kg/day. 

Conclusion:

Based on the abovementioned justifications, results from EC 270-608-0 OECD 422 study was determined to be adequateto fulfill the purposes of this endpoint.Therefore, NOAEL of 160 mg/kg/day for systemic toxicity (including neurobehavioral parameters) was proposed for EC 283-392-8, and use of read-across will eliminate the need for new animal testing while allow meeting the data requirements.

Applicant's summary and conclusion

Conclusions:
EC 283-392-8 has not been tested for reproduction toxicity, however experimental data on structurally related substances EC 270-608-0 was available and suitable for read-across.
Based on this study, NOAEL of 160 mg/kg/day for systemic toxicity (including neurobehavioral parameters) was proposed for EC 283-392-8.
Executive summary:

In a guideline repeated dose and reproduction / developmental screening study (OECD 422) conducted according to Good Laboratory Practices, WIL Research Labs (2010) evaluated the potential toxic effects ofphosphorodithioic acid, mixed O,O-bis(iso-Bu and pentyl) esters, zinc saltswhen administered to rats. This study was designed to evaluate the toxic effects, including neurobehavioral effects, of the test material to parental animals and to evaluate the potential to effect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition, and early postnatal development. The test material was administered orally by gavage once daily to 3 groups of Crl:CD(SD) rats at levels of 10, 40 or 160 mg/kg/day. The low- and mid-dose groups each consisted of 10 rats/sex and the high-dose group consisted of 15 rats/sex. A concurrent control group of 15 rats/sex received the vehicle, mineral oil USP, on a comparable regimen. Ten males/group selected for pairing were dosed for 14 days prior to mating through 1 day prior to euthanasia for a total of 28 doses. Ten females/group selected for pairing were dosed for 14 days prior to mating through lactation day 3 for a total of 40-52 doses; females that failed to deliver were dosed through the day prior to euthanasia (post-mating day 25) for a total of 40 doses. The extra 5 males and 5 females in the control and high-dose groups were not used for mating and were treated beginning on study day 0; following 28 doses for the males and 40 doses for the females, these animals were assigned to the post‑treatment period and remained on study for a 14-day non-dosing period.

Under the conditions of this screening study, no test material-related effects on reproductive performance, gestation length, parturition, reproductive organs, or neurobehavioral parameters were noted at any dosage level. Based on these results, a dosage level of 160 mg/kg/day was considered to be the no-observed-effect level (NOEL) for reproductive toxicity. Test item-related moribundity, clinical findings, and microscopic findings in the non glandular portion of the stomach, characterized by epithelial hyperplasia, hyperkeratosis, and inflammation, were observed in the 160 mg/kg/day group. The injury to the nonglandular portion of the stomach was localized and considered to be irritation from test item portal-of-entry effects. Based on these results, the NOEL for portal-of-entry effects was considered to be 40 mg/kg/day, and excluding the histologic injury to the nonglandular stomach, the no-observed-adverse-effect level (NOAEL) for systemic toxicity, including neurobehavioral parameters, was considered to be 160 mg/kg/day. In the absence of effects on the general physical condition of the F1 pups, the NOEL for neonatal toxicity was 160 mg/kg/day.